USPTO Patents Application 10551504

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(43) sis^mb

2005 M23 B (23.06.2005)

PCT

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WO 2005/056603 Al

(51) m^MPf-ftm. 7 : C07K 16/28, C12N 15/11,

1/15, 1/19, 1/21, 5/00, C12P 21/02, A61K 39/395, A61P
35/00, 35/02, 37/02, 43/00 // C12P 21/08

2478530

(21) Hl^aiMS^-:

(22) HUfsttiJH B:

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PCT/JP2004/018501

2004 *P12 ft 10 B (10.12.2004)

(30)

(71)

2003-415758

2003 ^12 ft 12 B (12.12.2003) JP

^SUgifekit^a (CHUGAI SEIYAKU KABUSHIKI
KAISHA) [JP/JP]; t 1158543 H»fMbE;¥Pil] 5 TS
5f 1^ Tokyo (JP).

(72) *3«fctf

(75) fiBj^/tbMA (-^iicoutro*;: i£$5

(KIMURA, Naoki) [JP/JP]; t 3004101 3Ste£!l&T;&S5
SfJ&«*# 1 5 3-2 i^ttttamaStt A IbaiaH
(JP). ±J1 (TSUCHIYA, Masayuki) [JP/JP]; t

4120038 s^pg»iaiiffiiftms6ip^ 1T113 sstsj^n

iC^art Shizuoka (JP). £ ;j£ (NANAMI,
Masahiko) [JP/JP]; t 4120038 ltra!lflNl|i8*§rtTS*lF1
1 Tl 1 3 SSife^nS^^ie^art Shizuoka(JP).
a flfc (TOMIMATSU, Takashi) [JP/JP]; t 1158543

Tokyo (JP). Jll-n IA (KAWAI, Shigeto) [JP/JP]; T

astt fa Kanagawa (JP).

(74) f^iA: 5f**Di£, ^(SHIMIZU, Hatsushi et al.); t
3000847 ^!l±;f mSlBT 1-1-1 IStfc-O < If tfjU

6 Pg Ibaraki (JP).

(81) f^CD&l^PSy, ^TCDS^roHrtffillA^

RTtg;: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR,
BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM,
DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, HR, HU,

ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS,
LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NA,
NI, NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE,
SG, SK, SL, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US,
UZ, VC, VN, YU, ZA, ZM, ZW.

(84) jisa («5*©&i^igy , £-TG>mm<n&m&m

ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA,
SD, SL, SZ, TZ, UG, ZM, ZW), 3.-7V7 (AM, AZ,
BY, KG, KZ, MD, RU, TJ, TM), 3 — □ V /i (AT, BE,
BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU,

IE, IS, IT, LT, LU, MC, NL, PL, PT, RO, SE, SI, SK, TR),
OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML,
MR, NE, SN, TD, TG).

— (54) Title: CELL DEATH INDUCING AGENT
■<tj (54) aa^5Elf»*iJ

i^s (57) Abstract: A DNA expression vector encoding 2D7sc(Fv)2, wherein the variable heavy chain (VH) domain sequence and the
variable light chain (VL) domain sequence of 2D7 antibody are linked together via 15mer linkers in the order of VH-VL- VH- VL,
is constructed. Then this vector is transferred into CHO cells to thereby establish a 2D7sc(Fv)2 production expression cell line.
2D7sc(Fv)2 expressed in this cell line is purified and subjected to a cell death induction experiment. As a result, it is clarified that

— _ 2D7sc(Fv)2 has an activity of concentration-dependently inducing cell death.

IT)

o (57) m#i: e> i*2D7tnH*a>£$Snr£Hi*iEyij (vh) t&m~^&mmwzm (vl) vH-vL-vH-vLcoati/ic

<S| => IC, ^^^*15mer©'J ^g^Lfc2D7sc(Fv)2*3— K-rSDNASSS^^^— *«^L,

0^-^CHOfflflS[C^ALT2D7sc(Fv)2^^^IJ$ffl^1*^^lZ:Lfco ttfflfl&*-C»ffl£#fc2D7w(Fv)2£«« U fflSS
5Eg§WHIfc£fT£ofc£: Z 2D7sc(Fv)2li;«Jtft#6<]lc$Hf§5E$-i§^"^".5);5 , 1t LTL^ C <t^?fl £>AMc£Eo

WO 2005/056603 1 PCT/JP2004/018501

m m »

[0001] fcftWte. HU&mWrt-%%Lft<Dsc(Fv)2Km't-Zo

[0002] HLA class ltfiMfe^ 3o©K^(a 1, a 2. a 3) 3&>£>7£545KD<D a §it, 12KD
© j3 2^n^n^y^(D-^^n^V^— fc£-?Xl&)$.&tiZ> 0 HLA^-<D^^§iJte

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x<d pjmm>^^tix^

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^rfcm HI^L-TV ^ yf « JVC I ^ Q
[0005] \C S thHLA class IA<D a 3VM>\Z.Mir&#ii$ 5H7 (##W^CitK5) ,

HLA class IA<D a 2K^^-^££l#:RE2 (##f^Citk6) fe&4kV>s<W£¥

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, ^sakavcv ^r^h— iy^(Dmmt^^fc<w^^4^(Dm^xh^tm

WO 2005/056603

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[0006] £i±<d£?k^ ^HLAm^Kx^mmmmmm, ms&zmmftm Km-rsm^fe

[0007] — ^2D7£iflcj^ th^rna— ^^^^Balb/c-^^^^SL-r^c^^^y^a—

[0008] &4b\ ^mil^P^^ra-rs^fStf^ff ^Sr^T^^-To

##f^Citfcl:Fayen et al., Int. Immunol 10: 1347-1358(1998)
##W3Cit£2:Genestier et al., Blood 90: 3629-3639 (1997)
##f^Ciffe3:Genestier et al., Blood 90: 726-735 (1997)
##f^Cit£4:Genestier et al., J. Biol. Chem. 273: 5060-5066 (1998)
##f^Cfifc5:Woodle et al., J. Immunol. 158: 2156-2164 (1997)
##f^Citfe6:Matsuoka et al., J. Exp. Med. 181: 2007-2015 (1995)
##W30tfe7:Goto, et al. Blood 84: 1922 (1994)

[0009] ^m®Mfe^(D£yfj;ytmzM.^X^tlfdb<DX&V^ ^z<D @ HLA class IA£r

i^&So f^ixjs. 2o(Dm^Br^fa^u ? 2o(DMBr^fa^'a-^, thfijfiu^
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[ooio] ^mm^n±mmm^m^ir^fc.m^mMm^n^fc. 0 2Di^m^ mm

^#m-f^^O^^^^J;^S#S*leukemia^Si-§^^^J;^#^tifc^^

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WKK^fTo/c(W02004/033499) 0 ^/c. ^HLA^#^Diabody/j^<Dig^^b£t

kfS, ^#w?£'l»_t#£*3^ ^^ImW^L/cMJI^ sc(Fv)2^-T3^^J;(9

mat, 2D7ffift<Dmm*i&w&mm (vfo w^bt^^e?ij (vd ^

VH-VL-VH-VL(D5lfetJ ? t^5 «t5 ^ ^tvetl^l5mer(Dy "Cg^Lfc
2D7sc(Fv)2?r=3— Ki-SDNA^ii-^^^— flE-^^— SrCHOjNHJ&lJl^A
LT2D7sc(Fv)2^^M«^WS:LfCo flM&*^^£i^c2D7sc(Fv)2^t

„ $^^2D7sc(Fv)2(Z)jfiL^^Stt^M-<Sfefe, ^^(Dlk^MSti^f^fL
td^^ 2D7diabody^i:b-<TiS^^jfiL^?||^0#rfg^®S§tLS^^^^ io f Co -

co^^ 2D7sc(fv)2«, ^^m^mm^^^xim^mm^m-m^u jfc
[oon] sp*,, &mmft. 2^(Dmm^m%mj&m^(Dmm^m%m?t^, a «

BIU WT(D [1] - [28] ^S#fci-5^»-Cfc3 0

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[3] 2o©m^^fJ«AtJ ? 2o(D^ Bj^fjj^^y^w-^ii^^v
Cl]^c«[2]^fE*(D^:#: 0

[4]y^— ^isT^y^-e&s^t^ftm^-rs, [3] ^ffiigcomf^o

[5] HLA^HLA class rCfc£, [1] — [4] GOV ^^fEfc^^L^o
[6] HLA class I^HLA-A-Cfc3, [5] ^§E<tO£rL#: o

WO 2005/056603

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0J££r£tfsc(Fv)2 o

[9]ia^lJ#^-:6. 7. 8^fE«(DT^/^ia^J^^^^CDRK 2. 3^^^^
^£r^t?sc(Fv)2 0

ClO]E^lJ#^-:3, 4, 5^fE«©T5y^ga?lJ^^^SCDRK 2, 3^fi-3S§|RT^
fSTOJ;I^WlJ#^-:6. 7. 8^fE*(DT^y^ia^J^^^SCDRU 2, 3^ff5g
^BT^®^Sfa-tfsc(Fv)2 0

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[12] ia»-!§-: 12^fE*©T^/^ga^J^'r§e^ RT^1W£r^i?sc(Fv)2 0
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[14] @a^lj#-^-: 14^|E^(DT5y^@B^J^i-5sc(Fv)2 0

[15] m$m^r: 2KmM<VT^/m®m&^'t&sc(Fv)2 0

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[20] [17]*fc«[18] ^lE«®^y^^^-K^«[19] ^fE*c(D-<^-^#

[21] £*T<£>X@£r^if [1] - [16] <DV>i*^^c:|Eife©*n:#:^li"t-S^'«fe„

(a) HLASr^fR-r5$n:^SrH®i*r5Xe

(b) (a)-e^Lfc^Lf*(Dia^J^i-. [l]-[16]»V^^^^fE«(Dfe ! L#:^=t— K

WO 2005/056603

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(d) (c)©^^-^i±MtiAt5Xg

(e) (d)<Dfe£Mti&&mmirZ>JM

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[25] [i^-{i6}(D\^irth^cmm<D^^^j]^tux^ir^^ mn&mmm

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[28] [l]-[l6]<DV>-f^^-?S*»^L#;^^^bT^1-§, SBMi

[0012] [BH]2D7i£^^-^Lf*(7)1#ia^i~KI"^fcSo EUAtt2D7diabodySr^U

diabody(HL5)«. 5merOy^7— "«^£;fxfcS§l oJ^ft«(VH) £<g§| oT^fft^C
vUtf*. ^#^^^J;t)— *^>fbi-5-^-e^$tt5o EUBtesc(Fv)2M^U
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^^W^^-^o #-eHo^^ia^J«5'#J^^TfjlJK^*EcoRI, BamHI, NotlO^O

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Sr^-f-ia'CfcSo (DfS 2D7 sc(Fv)2(D^/^i!i^n^b^A^m^- J r^b. (2)f3 2D7

WO 2005/056603 6 PCT/JP2004/018501

[g|4]in vitrolr*3{tS2D7 diabody£2D7 sc(Fv)2<Z>£0#3^p^?£tt»M:i^^i-|E|

[g|5]2D7 diabody£2D7 sc(Fv)2(Z)^J!^jt^t^ffjlJ^tt(Di:l:K^i-BI'efc5o
ra6]^J^^4»a2D7sc(Fv)2:&^

[0013] #DSBI!f2. 2o<Dm®BT^fB«^tK2o(D«BT^fB«^^, tbfi«fetlg

mm^mmmim. mmmmmmim. mmwmmim. mmttmmmt
[ooi4] Mfamm&ftm, mmmmmmm^<D±mi^m(DM^t^mmmm^m

Lfc^fif jfrt^S^Cperipheral blood mononuclear cell, PBMC)) , v^HJ^Jfcif

[0015] —&mz. ^S^HLA^f^i*^^^fi^tt^i-^^^igG^#:^^-e^

[0016] ^K<D^<*:^^jjiHJ^^^bT^^^fl^-rSd^^*, JurkatlHJ§&XJ3:

WO 2005/056603

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[0017] fc*3V ^T^i, _tfE. 2oom® rT^0J&& t^2o©gi R^ifi^^. t

[0018] IJ1*3V vC, HLA^J3 N th jfc.^^LHC^s5fei~5 0 HLA^H^classI^classII

i-^ffi$^L. classI^LTiSHLA-A, B, C, E, F, G, H, }t£¥&%RtbtlT3sQ^ classll

HLA^Xhtim^fKU^f^^^, tft^<nclasslKftM£tl%ft^-XhiQ, X
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[0020] sc(Fv)2«. 2o©fi BT^fg^ ( [VH] ) ,K.TJ*2o<Dig^ ( [VL] ) ^

— #'C|^'^LT^^:^^y-<y 0 ^-K^bfcfe ! i:#:'efe§ (Hudson et al, J Immunol.

WO 2005/056603 8 PCT/JP2004/018501

Methods 1999 ; 231 : 177-189) 0 sc(Fv)2f±^ 2^><DscFv(^>'^/l^f-^f^Fv)
(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883,
Plickthun TThe Pharmacology of Monoclonal AntibodiesJ Vol.113, Resenburg IfiTJ
Mooreli, Springer Verlag, New York, pp.269-315, (1994))£ry>>&— ftM^

m*&<DMm%mz.mM£ti'r, }f<Dx^j:immx^ht^x\^xhx<, m«\

[VH] [VL] V>%— [VH] V>%— [VL]

[vl] T-y^i— [vh] [vh] [vl]
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[vl] y [vh] Tstt— [vl] y^— [vh]

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e^i~§sc(Fv)2-efe§ 0
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X^% 0

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Ser

Gly • Ser

GlyGlySer

Ser "Gly Gly

Gly • Gly • Gly • Ser

Ser • Gly • Gly • Gly

Gly • Gly • Gly • Gly • Ser

Ser • Gly • Gly • Gly • Gly

Gly • Gly • Gly • Gly • Gly • Ser

Ser • Gly • Gly • Gly • Gly • Gly

Gly • Gly • Gly • Gly • Gly Gly • S er

Ser • Gly • Gly • Gly • Gly • Gly • Gly

(Gly • Gly • Gly • Gly • Ser)n

(Ser • Gly • Gly • Gly • Gly)n

[0025] ■^■mtmrnv^—at^mmm)^ ^^(DmmKmi%m^thx\,^mm

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[oo26] 4-D(Dm^mM^w^i-^>m^a^ mig. 3o<Dy^#— ^^^5^ ^

[0027] ^^Cfel^T, #?3;U^sc(FV)2(D»LT«, KT®(a)- (i)<DV^-f jft^fcfBife

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(a) m$m^r:3^ 4, 5^fEiSc<Z)T^/^ia^ib&3CDRl, 2. 3^rr3M#^^
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sc(FV)2C0T^7BIBa^ISrBH^J#-^ : 2 ^^i~ 0
[0029] Ha^!l#-^: 2^|B*fe<DT^y^Ba^!lSr^r*t"5sc(Fv)2*fctt, BB?!I#-J§-: 2fc!B

WO 2005/056603

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fetHCtt^jfiT^^^i:^^ g 6«j£l/CfcMb (Humanized) , (Chimeric) {bbT

So

[0030] ^-eratgw^i^^j xmttez&imK mpm^:2Ki&M<Dmm&ftir
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[0031] feS^y^^KMf^^^^l^^y^^K^MMi-Sfcfe®, ^il#^J;<£n

^M^Xhtll^, §P^#MW^MfHS^(Hashimoto-Gotoh, T. et al. (1995) Gene
152, 271-275. Zoller, MJ, and Smith, M.(1983) Methods Enzymol. 100, 468-500,
Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz
HJ(1987) Methods. Enzymol. 154, 350-367, Kunkel,TA(1985) Proc Natl Acad Sci
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[0032] mmir^T^ymwcm^Kmm^ti^^K m^, 3or^ym^xm, 0*l<
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h, k, s, t) , mffiiMwm&ftirzT^smiG, a, v, l, i, p) , 7km^mmm&^
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xfTK^miMm&^i-&T^ym(D^ n, e, Q),mm^wm&&1rZT$;&t(.R

, k, h) , ^M^{Mm&^~r%T^ym(H, f, y, w) &m?z^k&~?%% (»

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tl/CV ^5 (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666 ,
Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500 , Wang, A.
et al., Science 224, 1431-1433 , Dalbadie-McFarland, G. et al., Proc. Natl. Acad.
Sci. USA (1982) 79, 6409-6413) 0

[0033] &?&w<D&cma^ &ftm<Dttifc<D7$y&mmzw&i®<D7$y&&m&ttM
^m^^^xn^v^-y^tvxn, flag(h opp , t. p. et ai.,

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l l -±Z.(DftmZ-^^Xir'e\C$UiL£frlX\^ (#|xif^ US5057313, US5156840) o ^

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_h<D|^— ttSr^t-o ^y^^^K^^^tt^S-r^^^, JCj» (Wilbur, W. J. and
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[0038] ^^K(Dsc(Fv)2^^H#i^^n(D^i^J;(9f^®-rS^^^-e^§ o 0tJ&tf. HLA

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Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496 ; Pluckthun,

A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515 ; Lamoyi, E., Methods
Enzymol. (1986) 121, 652-663 ; Rousseaux, J. et al., Methods Enzymol. (1986) 121,

663-669 ; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137#

[0039] HLA^Mff5i-3#L#wia^j«. mzte&(Dffifo(Dmp\&F8^zzk&^m~e&> i ().
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m^ri^fc 0 2D7sc(Fv)2(D^ia^j (m^m^r: i) *3<£^T^mia^j (ia^j#^-

:2)^g|2^-r o

[0077] [Hife{?!j2] 2D7sc(Fv)2m&ft&$&ti&W<Dffifi.

PvuI-e-gJifbli:ffi^L/cpCXND3-2D7sc(Fv)2 20 n g^CHO|0Ji^ (DG44^) i-JU
T^i^^electroporation^fet^jC^^Ab/to

[0078] CHO-S-SFM-IIigifi C-f ^tf r-nv 5 ^) 7;^#LfcDG44£BJteice-cold PBS"C2[U
gfe#L^lX10 7 /ml^^§J;5^PBS^«L^o ^TU-20 /z g©_hfE^^K^M

m^V^(1.5KV, 25 ii FD) ^r-^-X.7t 0 S^#J^«^^L96 well
plate^BJ^Jf^^ iMS500 ,u g/ml<D G418OfVfc->Pv^>0 Sr^ODLfc
CHO-S-8FM-IIi#J*cp-ej##^To^ 0 4fLfcf-3n^- ^|tJ30^n— ^5iffc°
y^Ty^U ^n^J§#J^?f 4 3 »2D7sc(Fv)2(D^^a^FLAG^L#;(^V) ^ffl
Wcr>3i;^:/;7^H-J;9fS^c 0 ||tl§?S<7)ri^o/c^n— >^5nM MTX^fr
S^y— (DCHO-S-SFM Iligife (/fVlfhn^ni^-eJg^^TV^, i#^.x^—

[0079] [Hifef?!l3]2D7sc(Fv)2(D^:iltM

T-125^9^^'Ci^^^^ 7 /^^hO2D7sc(Fv)2r^0^CHO|0JJ^^lXlO 5 /mnc
ftSJ:?^— #>/V (CHO-S-SFM Iligift 250ml/#h/V)^#L/c o 37°C-Ci#^
U 6 0 mt-J§»^0i|2bfe o ^>^^J;o-C^E^^^^L^m, 0.45 M m7>f;^

[0080] 2D7sc(Fv)2(D^fM«J^TO^*5(9tfo^ 0

^-f\ buffer A (20mM Na-phosphate pH6.8) ^^S^bbfcHydroxyapatite ^7-A(
micro prep ceramic Hydroxyapatite type I, Bio-Rad) f£p3^l^7ti$il| Jr?ff^applyL-
fc 0 buffer AW^i^$fc#Ufc^ buffer C (250mM Na-phosphate pH6.8)-C
2D7sc(Fv)2Sr^mbfc 0 2D7sc(Fv)2Sr-a-tf^9^^3^?r#^(Dbuffer A"C^r^bfc
^ ^^^r Anti-Flag M2T^fn— 7-77^^T^7^ (Bio-Rad) ^applyb7t 0 Z-(Dl3

WO 2005/056603

26

PCT/JP2004/018501

5^£rbuffer C (50mM Tris-HCl pH7.4, 150mM NaCl, 0.01% Tween 20)-C#S#'Ufc

buffer D (lOOmM Glycine H3.5, 0.01% Tween 20)^2D7sc(Fv)2^f§mU/c o HWX
L7ci^^/V»[i:^^^|giS25mMt^5J;5 ^Tris-HCl pH8.0-C^PL/c o -?:<£> ^
, ^(D^^^a^. ir^hyyi^^^YM-10 (AMICON) "CigSBgU Superdex200HR
(26/60)#^A (7v^tA7r/W^T) ^iSy/^ai^n^h^AW^l^fflL^:

o

0.01% Tween 20^frPBS^y/V^M^P^b^Aff$^TV \ ^V^V^mHf

6t#fc£, ^®J3^^^»^52Kd (Dfug^Oit 0 — ^^U03(l)»,
|^C<52Kd-e^t±i$HS2D7 diabodyOt 0 — ®:b^(g|3 (2) #flg) 0
r^fe»W-C#|gL/c2D7sc(Fv)2te. g ift^^EllB^-tlflit (l«t

2D7sc(Fv)2S &mffatl,fz. 0 W^XAz.^-^ JV(D— %\i&SDSm,M<$KW)&£XF$&%k&
lfi^^D R pttir^by7V5/^°YM-10 (AMICON) -C^fitU 2D7sc(Fv)2ffiS^ B B ptbT

[0081] C^ifef?!j4] 2D7sc(Fv)2(D|0JJ^^Ef|3g?g'|4(D|BlJS

th#MM$BJ!ftttARH777jtt^ 10% FCS^tpRPMI1640j§ifi(^Vtfha^aiV)
"CI X 10 5 cells/well \Z.1£Z> X 5^24 well platefcjjfflJ&Stft^fCo wtL^, ^f^Lfe
2D7sc(Fv)25r|#2iJSl00ng/mL 250ng/ml^^SJ;5^^Pb/Co ^fcJfctJM&flc

i:LT, Jfil®lLfc2D7diabody(HL5)SrSU<DweU^|^— ^#-e^JPLfCo 37°CC3H#5a

#*flJ&£rlelJRU Pim0i(5 v g/ml PI, 2% FCS/ PBS) fc*BJ&£r®$i®Ufco
)i3tL"C^?l.-C15^>'^^<— YUtM., flow cytometory^fflV^-CPI-C^fe^^c
^Bflfr<D§iJ^^pJ5£L-/c (EPICS ELITE, COULTER) 0

[0082] 2D7sc(Fv)2^*m^#Wi-lHJi^^Srfl^»-rS^tt^L-TV^S^i^^
Wk/^tOfeofco ^fc-^<D?g-'|4te, linker 5mer^VH^VL^o/£V ^£2D7diabody (

WO 2005/056603

27

PCT/JP2004/018501

HL5) tmtm^^/^-h^t^M^t^ Kl±CO^^XK>, in vitroi^ftS&BJI&^E
m^^^Km^XU, 2D7sc(Fv)2f3:2D7diabody(HL5) tmWi^^^XV

[0083] [HJfe0!l5] 2D7sc(Fv)2<DfflJ&*t^f&Mt£<^5£

thEBV-transformed B^^^IM9^BJi^(ATCC) ^3X10 3 cells/well7:\ ^/cth^^—
^5/MJV/^MS5fe^0J9^HS-Sultan^Ji^^lXlO 4 cells/well^&3i;5^10% FCS^
tpRPMI1640i#ii-e^rf^L, 96 well plate i^Ji:^7c 0 ^tL^, $^L7c2D7sc(Fv)2 „
*3J;TX 2D7diabody(HL5)?f|#liS0, 0.0032. 0.016, 0.08, 0.4, 2 m g/mlt£ft5J;5
KSfe&PU 37°CCi:«b7t 0 3 0 f^Ml. l0^WlJSWST-8dr5/h(^fr^^)^

-^<Df£JH N 2D7sc(Fv)2te|§S#c#lftiaM9;Mft. HS-SultanlBJS&Oit?®^
2D7 diabody^|al#(D|0JJ^jf ^K!!j^tt^#0^^^^>^, 0 f Co

[0084] [HJfef?!j6] MtMmMWi2D7sc(Fv)2 *3 j;tJ«»t'|4^^2D7diabody(HL5)(D|iM

250 mmol/L NaCl^I^Tween 20 0.05 vol%-a^20 mmol/Ly^^HWIR (pH 7.0).
Na 125 I^«U?2D7sc(Fv)2^fc^2D7diabody(HL5)^IS ; ^^Bbfc 0 0. 15 mol/L
NaCl^|fe^^M^M$^:^«(5X5 Dim)^/^ #7^_t^<D-^, ^>jjft^
32 mg/mL Chloramine T}§^l&^3^ ^>*S^[^f|ij^^§ j;^^^;/^— ^f^^

[0085] ^M-C55>R9$tgbfc|£, §rfc^32 mg/mL Chloramine Tj§t^^iA^fc6IS

fcfoWKTween 20 0.05 vol%-Q-^PBS(-)^^P#, fcjfcm&Tween 20 0.05 vol%
-a-WPBS(-)-C^P^t:U/cPD-10 column (7vi/-V^77;Wi/7) t£<£Hi\ Tween 20
0.05 vol%-a-^"PBS(-)^T^Hi$**MJ^<^ 125 I^^^b/to Superdex200

10/300GL#5^ (7v^wr;w7) ^J^y/i^M^u Sfc£M£3— K^lfi

2D7sc(Fv)2 jSXXfW&O&a— K^^2D7diabody(HL5)^f«L7C 0

WO 2005/056603

28

PCT/JP2004/018501

[0086] C^Jfef?!j7]^^^^ltm^^2D7sc(Fv)2^j:^^lttt2D7diabody(HL5)^i|itni^

Jttt^^(C.B-17/Icr Scid JcU B^WT) ^$C#ttttJf!i2D7 sc(Fv)2 $$£XfiitC
#tttt5It2D7diabody(HL5)^l mg/5 MBq/kgT^SMfNJRrtix^Ucio i£^15, 30

u 2, 4, 8, 24B#r B m^^-^/^ffi !i T^^^^Hib, <t?m£<9^<y>mmis

12,000 rpnTC5;£f^ja^U jfil#^iib/c 0

*&^t^££^mufco ^f^PJ^, jfiL«I^^W3|5i7K*3J;OTCA 25 w/v%

^SriP^L. 3t##, 4 o C-C3000 rpm, lOfrm&bfrffiUZo _h?f ^T^tV-^—
[0087] ^|^'|4^m2D7sc(Fv)2 *5j;^lttt^^2D7diabody(HL5)^^-|¥^^i&.#^

TCAttmm / ^McMmm&nr.jfm'CM^fc.m6) 0 ^tt^m2D7sc(Fv)2 w&

tB»¥^M«^tL^tL2.30H#F H 1^J;t^l.64H#FHl-C2D7sc(Fv)2«2D7diabody(HL5)J;
[0088] HLA class IA^fgf^^ig^^Lf* (diabody) ^sc(Fv)2^^«}tB^^i-Sr £ K X

WO 2005/056603

29

PCT/JP2004/018501

m^comm

[2] 2^<DMm^mnmRxf2-DCDmmpjmnmfrK —^m^v^^^-N^m^
[4] v^*—&i57$ym~?hz?ik&&wtbir^ m^.m3Kmm(D^ii$. 0

[5] HLA^HLA class rCfc£, fi5j<£gl~4<Z)V^f ^^fE^^^lf^o
[6] HLA class I^HLA-A-CfcS, ft^^SS^fEftC)^^
[7] sc(Fv)2-e&§ff5jc3Sl— 6(Dl ^frtf^fEifc^Sntffc,

[8] ia^iJ#^-: 3 N 4, 5^fE*c<DT^/liia^J£^£CDRl, 2, 3£rirr£M Rl^fS^
£r^t?sc(Fv)2 0

[9] ia^ij#^-:6, 7, 8triB4fe<DT^y^ga^id^3fc5CDRi, 2, z^i-zmmnmnu

£r^tfsc(Fv)2 0

[10] @B^IJ#^-: 3, 4, 5l£|B4!6<£T^yg£Sa^ja>£>ft5CDRl, 2 N 3^PrT5M

*3«ta ? Ba^!i#-^:6. 7. 8^iBife<oT^y^Ba^id^5fe5CDRi. 2, 3^-rse^RT

^^££r^t?sc(Fv)2 0
[11] BB^J#^: 10tr|B4fe<DT^^Ba^J«r^Ti-5a^ BT^^^Srg-tfsc(Fv)2 0
[12] IE^IJ#^-: 12^fEife(DT^y^ia^J^Wr5M "ST^i&Srg-tf sc(Fv)2 0
[13] @a^ij#^-: 10^|E*(DT5y^@a^J^-rsm^ bT^^«*5J;^WJ#^-: 12^

fE«®T^y^ia^j^#i-§M'5r^fM^^'a-tpsc(Fv)2 0

[14] ia^lj#^- : 14 ^fE*OT^y|iia^J^'r§sc(Fv)2 0
[15] ia^ij#^: 2Kffim<D7$smmm&j?irZ>sc(Fv)2 0

[16] ff^3S8— 1 5<Z)V tLfrlZ.mM<7)7^yMMF\ t-*3V ^TlXM^T^/^Sg
fat ^l^(D^tt^^-r§sc(Fv)2 0

WO 2005/056603

30

PCT/JP2004/018501

[17] fiMfc®l~ 16»v^^^^fE*»^tf*^— Ki-§*°y^^-^K 0

[is] it ^iii7^ts*(Z)^y^i/^K^hyv^vh^#T^vN/r^y^vxL,

[19] M^l7*^if*l8^|B*fe©^!J^^^KSrg-t?^^— o

[20] ff^l7^c«18^fE*®^°y^l/^K^c«ft^lI19^fE*©-<^— ^
[21] J^T»X@^tfff^iSl-16(Z)V>T^^-fE^(D^f*^Mi-S^ 0

(b) (a)-ef«b^#:(Dia^J^^, ft^IIl-16(DV^^^-fE*0^rL#:^

— K-rs^y^^w^K^M-rax@

( c ) ^(D^y^^^^K^^^^^— &{^mir%jm

(d) (c)<D-<^— ^±!0tet^At5Xg

(e) (d)(D^^|0JJ^^j##-r§X@

[22] mM^i—i6<D^thfrte^M<D&ft&&&&frbL,xi£ftir^ m^mmMo

[25] ft^3Sl-16®V^T^^^?E*-Z)^:#:^gi^^b-C^«, M^it^ffjiJ
Mo

[26] — 16©V>i*^^C|B4fe©iii#:Sr^r^l^^bT^-rS. MMiJo

[27] mm&fammmxh%mMm26KmM<Dttmmm 0

[28] W^l — 16©V^^^^|B*fe©^Sr^^i:L"C^-r5^ IBMM

1/5

WO 2005/056603

2/5

PCT/JP2004/018501

[02]

10 20 30 40 50 60 70 80 90 100

CCTgaUgcaCCATGCGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAATAACTGCAGGTGTCCATTGCCAGGTCCAGTTGCAGCAGTCTGGACCTGAG
MRWSIYIFLFLLS/TAGVHC§VQLQQS&?£
110 120 130 140 150 160 170 180 190 200

CTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCTTGTAAGGCTTCTGGCTACACCTTCACAGACTACTTTATACACTGGGTGAAACAGAGGCCTGGACAGG
LVKPGASVKMSCKASGYTFTDYF I HWVKQRPGQG
210 220 230 240 250 260 270 280 290 300

GACTTGAATGGATTGGATGGATTTTTCCTGGAGATGATACTACTGATTACAATGAGAAGTTCAGGGGCAAGACCACACTGACTGCAGACAAATCCTCCAG
LEW I GW I FPGDDTTDYNEKFRGKTTLTADKSSS
310 320 330 340 350 360 370 380 390 400

CACAGCCTAGATTTTGCTCAGCAGCCTGACCTCTGAGGACTCTGCGATGTATTTCTGTGTAAGGAGTGACGACTTTGACTACTGGGGCCAGGGCACCAGT
TAY I LLSSLTSEDSAMYFCVRSDDFDYWGQGTT

410 420 430 440 450 460 470 480 490 500

CTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggaagcCAAATTGTTCTCACCCAGTCGCCAGCAATCATGTCTGCAT
L T V S S GGGGSGGGGSGGGGS QIVLTQSPAINISAS
510 520 530 540 550 560 570 580 590 600

CTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGGACTGGTTCCAGCAGAAGCCAGGCACTTTTCCCAAACTCTGGAT
PGEKVTITCSASSSVSYMHWFQQKPGTFPKLWI
610 620 630 640 650 660 670 680 690 700

TTATAGCACATCCAACCTGGCTTCTGGAGTCCCTACTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAA
YSTSNLASGVPTRFSGSGSGTSYSLT I SRMEAE

710 720 730 740 750 760 770 780 790 800

GATGCTGCCACTTATTACTGCCAGCAAAGGACGAGTTATCGACCCACGTTCGGCTCGGGGAGAAAGTTGGAGATAAAAggaggtggtggcagtggtggcg
DAATYYCQQRTSYPPTFGSGTKLE I K GGGGSGGG

810 820 830 840 850 860 870 880 890 900

gc fegatcc| ggtggcggtggctcaCAGGTCCAGTTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCTTGTAAGGCTTCTGG
G S G G G G S OVQLOQSGPELVKPGASVKMSCKASG
910 920 930 940 950 960 970 980 990 1000

CTACACCTTCACAGACTACTTTATACACTGGGTGAAACAGAGGCCTGGACAGGGACTTGAATGGATTGGATGGATTTTTCCTGGAGATGATACTACTGAT
YTFTDYF I HWVKQRPGQGLEW I GWI FPGDDTTD

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100

TACAATGAGAAGTTCAGGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATTTTGCTCAGCAGCCTGACCTCTGAGGACTCTGCGA
YNEKFRGKTTLTADKSSSTAYILLSSLTSEDSAM
1110 1120 1130 1140 1150 1160 1170 1180 1190 1200

TGTATTTCTGTGTAAGGAGTGACGACTTTGACTACTGGGGCCAGGGCACCACTCTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctgg
YFCVRSDDFDYWGOGTTLTVSS GGGGSGGGGSG
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300
cggtggcggaagcCAAATTGTTCTCACCCAGTCGCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTA
G G G S 0 I VLTOSPA I MSASPGEKVT I TCSASSSV
1310 1320 1330 1340 1350 1360 1370 1380 1390 1400

AGTTACATGGACTGGTTCCAGCAGAAGCCAGGCACTTTTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTACTCGCTTCAGTG
SYMHWFQQKPGTFPKLW I YSTSNLASGVPTRFSG
1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
GCAGTGGATGTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGACGAGTTATCCACGCAC
SGSGTSYSLT I SRMEAEDAATYYCQQRTSYPPT
1510 1520 1530 1540 1550 1560 1570 1580

GTTCGGCTCGGGGACAAAGTTGGAGATAAAAgactacaaggatgacgacgataagtgataaicIIccIclaat
FGSGTKLE I KDYKDDDDK**

WO 2005/056603

3/5

PCT/JP2004/018501

[03]

(1) 2D7sc(Fv)2

mAU r i0 - 1 3

150

100

(2) 2D7 diabody

mAU
600

[04]

O"-

■Mil

G

4fE3

2D7 sc(Fv)2

200. 43

□ 0

■ 100ng/ml
H250ng/ml

diabody (HL5)

sc(Fv)2

WO 2005/056603

4/5

PCT/JP2004/018501

[05]

120
^ 100

G 60

&

£3 40
*H 20
0

IM9

0.0032 0.016 0.08 0.4

HS-Sultan

120

o 100

0 s *

80

■fifln

G 60

i§3 40

*H 20

0

0.0032 0.016 0.08 0.4

WO 2005/056603

5/5

PCT/JP2004/018501

INTERNATIONAL SEARCH REPORT

International application No.

PCT/JP2004/018501

A. CLASSIFICATION OF SUBJECT MATTER
Int. CI 7 C07K16/28, C12N15/11, 1/15, 1/19, 1/21, 5/00, C12P21/02,

A61K39/395, A61P35/00, 35/02, 37/02, 43/00//C12P21/08

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

Int. CI 7 C07K16/28, C12N15/11, 1/15, 1/19, 1/21, 5/00, C12P21/02,
A61K39/395, A61P35/00, 35/02, 37/02, 43/00//C12P21/08

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
CAPlus(STN), MEDLINE (STN) , BIOSIS (STN) , WPIDS(STN), JICST FILE(JOIS)
Swiss Prot/PIR/GeneSeq, Genbank/EMBL/DDBJ/GeneSeq

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

P,X

BRUENKE J. et al., "A recombinant bispecific
single-chain Fv antibody against HLA class
II and FcyRIH (CD16) triggers effective lysis
of lymphoma cells", British Journal of
Haematology, April 2004, Vol.125, No. 2,
pages 167 to 179

SEKIMOTO E. et al., "A recombinant HLA Class
I-Specific Single Fv Diabody Induces Cell
Death in Human Lymphoid Malignancies.",
Blood, November, 2003, Vol.102, No. 11,
page 933a, Abstract. #3474

1-7, 17-2E

1-14,16-2!

EE] Further documents are listed in the continuation of Box C.

1 1 See patent family annex.

* Special categories of cited documents:

"A" document defining the general state of the art which is not considered
to be of particular relevance

"E" earlier application or patent but published on or after the international
filing date

"L" document which may throw doubts on priority claim(s) or which is
cited to establish the publication date of another citation or other
special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or other means

"P" document published prior to the international filing date but later than the
priority date claimed

"T" later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
the principle or theory underlying the invention

"X" document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive
step when the document is taken alone

"Y" document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art

"&" document member of the same patent family

Date of the actual completion of the international search
11 March, 2005 (11.03.05)

Date of mailing of the international search report
29 March, 2005 (29.03.05)

Name and mailing address of the ISA/

Japanese Patent Office

Authorized officer

Facsimile No.

Telephone No.

Form PCT/ISA/210 (second sheet) (January 2004)

INTERNATIONAL SEARCH REPORT

International application No.

PCT/ JP2004/018501

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

P,A

KIPRIYANOV SM. et al . , "Effect of domain order
on the activity of bacterially produced
bispecific single-chain Fv antibodies",
J.Mol.Biol. , 2003, June, Vol.330, No.l,
pages 99 to 111

Laurent Genestier, et al . , "Fas-Independent
Apoptosis of Activated T Cells Induced by
Antibodies to the HLA Class I 1 Domain",
Blood, 1997, Vol.90, No. 9, pages 3629 to 3639

Hudson PJ. et al . , "High avidity scFv
multimers: diabodies and triabodies . " ,
J. Immunol .Methods . , 1999, Vol.231, No. 1-2,
p. 177-89., Review

JP 7-503622 A (The Dow Chemical Co.),
20 April, 1995 (20.04.95),
Full description

& WO 94/13806 Al & EP 628078 A

S US 5877291 Al

CO MS . , et al., "A humanized antibody
specific for the platelet integrin
gpIIb/IIIa." J. Immunol., 1994, Vol.152,
No. 6, p. 2968-76.

WO 2004/033499 Al (Chugai Pharmaceutical
Co., Ltd. ) ,

22 April, 2004 (22.04.04),
(Family: none)

1-14, 16-2*

1-7,17-2!

1-7, 17-2!

1-7, 17-2*

1-14, 16-25

1-2!

Form PCT/ISA/210 (continuation of second sheet) (January 2004)

mtamm^-w pct/jp 2 0 0 4/0 1 8 5 0 1

a. ^rog-ra^^g (mewf^sc (ipo )

Int. CI. 7 C07K16/28, C12N15/11, 1/15, 1/19, 1/21, 5/00, C12P21/02, A61K39/395, A61P35/00, 35/02, 37/02, 43/00
// C12P21/08

b. n^&ftotz-frm

m^*ftofrm'Mmn mmm^m (ipo )

Int. Cl. 7 C07K16/28, C12N15/11, 1/15, 1/19, 1/21, 5/00, C12P21/02, A61K39/395, A61P35/00, 35/02, 37/02, 43/00
// C12P21/08

CAPlus(STN) MEDLINE (STN) BIOSIS(STN) WPIDS(STN) JICST7t^(J0IS)
SwissProt/PIR/GeneSeq Genbank/EMBL/DDBJ/GeneSeq

C.

PX

BRUENKE J. et. al. , "A recombinant bispecif ic single-chain Fv
antibody against HLA class II and Fc y RIII (CD16) triggers
effective lysis of lymphoma cells"

British Journal of Haematology, April 2004, Vol. 125, No. 2, p. 167-179

SEKIM0T0 E. et. al. , " k recombinant HLA Class I-Specif ic Single Fv
Diabody Induces Cell Death in Human Lymphoid Malignancies. "
Blood, November 2003, Vol. 102, No. 11, p. 933a, Abstract. #3474

1-7, 17-28

1-14, 16-28

[x] cm<om^%-xw^mm^x\^.

tej mmmmnM<Dtiim±fcte¥?m : xib%tiK mmmmn
rpj brums hw-^ fr~om%im<D±McoMmtfj:z>mm

r&j isi-^Tyf7r?9-X»

11. 03. 2005

mm<m&<»m& 29, 3. 2005

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KIPRIYANOV SM, et. al. . "Effect of domain order on the activity of
bacterially produced bispecific single-chain Fv antibodies. "
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triabodies. "

J Immunol Methods. , 1999, Vol. 231, No. 1-2, p. 177-89. Review.

JP 7-503622 ACT 9*9 KM =-) 1995. 04. 20, m'm^±M
& W0 94/13806 Al & EP 628078 A & US 5877291 Al

CO MS, et. al. , "A humanized antibody specific for the platelet integrin
gpIIb/IIIa. "J Immunol. , 1994, Vol. 152, No. 6, p. 2968-76.-

W0 2004/033499 Al ( WSaitfcjS^fcfc) 2004. 04. 22

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